O-trimethylsilylquinoxalinol derivatives of aromatic α-keto acids : Mass spectra and quantitative gas chromatography

As an extension of earlier work on aliphatic α-keto acids, a method is described for the quantitative gas chromatographic determination of urinary aromatic α-keto acids. The keto acids are derivatized with o-phenylenediamine to yield the quinoxalinols. These compounds are chromatographed after trimethylsilyation.The aromatic keto acids are stabilized by sodium dithionite (4 mg/ml urine) and storage below 0°. The final derivatives are stable for weeks at room temperature.Low resolution mass spectra are reported. The fragmentation mechanims are elucidated by analysis of O-trimethylsilyl-(TMS)-quinoxalinols, O-(TMS-d₉)-quinoxalinols and O-TMS-6(7)-chloroquinoxalinols.     

A Spectrophotometric Microdetermination of Keto Acids with 3-Methyl-2-benzothiazolone Hydrazone

Various keto acids react sensitively with 3-methyl-2-benzothiazolone hydrazone to form the azine derivatives, which give the characteristic ultraviolet absorption spectra. The effect of pH, temperature, reaction period, buffer and the concentration of the reagent on the reaction and the spectra were investigated. Optimal conditions for the microdetermination of keto acids were established. Microamounts of keto acids (0.02 ~0.3 or 0,4 μmole) were determined rapidly by this simple method with satisfactory results. Amino acids, fatty acids and most inorganic salts did not interfere with the determination of keto acids.     

Separation and determination of 14C-labelled intermediates of the citric acid cycle and related compounds

A new systematic procedure is presented which permits a complete chromatographic resolution of the intermediates of the citric acid cycle as well as the related amino and keto acids. The adsorption onto ion-exchange columns followed by the subsequent elution therefrom forms the first step of the present procedure. Meanwhile, unstable keto acids are enzymatically converted into the respective amino acids. This preliminary process is very effective in desalting the biological specimen and also results in a broad division of a large number of intermediates into three subgroups, i.e., amino, keto and other organic acid fractions. Each of these subgroups is then readily resolved into individual acids by means of one-dimensional thinlayer chromatography. ¹⁴C-Compounds added to the reaction mixture of rat liver mitochondria were recovered with a good reproducibility throughout the entire course of the present procedure.     

Diet treatment of branched chain ketoaciduria studied by proton magnetic resonance spectroscopy

Summary A novel nuclear magnetic resonance method is proposed for the diagnosis and follow-up of patients affected by branched chain ketoaciduria. The method allows quantitation of the branched chain amino acids (BCAA's) such as leucine, isoleucine and valine and of related keto- and hydroxy acids by means of a single spectrum. The method implies short time of analysis, as opposed to the very long time required by the techniques currently in use (amino acid analyzer combined with gaschromatography/mass spectrometry of keto- and hydroxyacids), it is easy and suitable for adjustements of the dietary treatment even on a daily basis. The case of a 15 days old newborn child, presenting muscular hypertonicity was unambiguously diagnosed in few minutes by means of one single NMR spectrum of urine. More interestingly, NMR spectra of serum in the following days were suitable for quantitating amino-, and keto acids as well as other metabolites of relevance in the follow up of the dietary treatment of the disease. After a diet lacking of BCAA's, to eliminate keto acids, a low BCAA diet was introduced, that succeeded in keeping the serum levels of the three amino acids within the normal range, while dropping the related keto acids.     

Reversed-phase high-performance liquid chromatographic analysis of branched-chain keto acid hydrazone derivatives: optimization of techniques and application to branched-chain keto acid balance studies across the forearm

A sensitive method of quantifying branched-chain keto acids in plasma and whole blood samples is described. It is based on the separation by ion-pair reversed-phase liquid chromatography of 2,4-dinitrophenylhydrazine derivatives with ultraviolet detection. The sample clean-up steps that are usually required for reversed-phase high-performance liquid chromatography are eliminated. A reduction in ketoisocaproate isomer formation is obtained by incubation of derivatives in ice. The method is reproducible (coefficient of variation 2%, n = 5, at the 200-pmol level) and the ultraviolet response is linearly related to branched-chain keto acid concentration. Recoveries are high (>95%). Other keto acids do not co-elute with branched-chain keto acids. Because of its sensitivity and precision, this method can be proposed for whole blood branched-chain keto acid balance studies across organs.     

Formation of keto acids by barotolerant marine bacteria under atmospheric pressure]

Production of keto acids by washed cells of the barotolerant strains was studied under atmospheric pressure on a glucose-mineral medium in balloons made of the copolymer of ethylene with propylene. Production of keto acids in these conditions requires elevated concentrations of glucose and minimum concentrations of ammonium nitrogen. Active aeration inhibits the accumulation of keto acids by the barotolerant bacteria. The majority of the barotolerant bacteria did not form free extracellular keto acids, other bacteria liberated comparatively small amounts of these acids. The ability to accumulate keto acids was different in particular strains.     

Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.     

New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.     

Determination of L- and D-amino acids in foodstuffs by coupling of high-performance liquid chromatography with enzyme reactors

Summary. A technique is described for the enantiomeric determination of L- and D-amino acids. It works on the principle that the separation efficiency of high-performance liquid chromatography is coupled with the specificity of enzymes and the sensitivity of electrochemical detection. After separation on a lithium cation-exchange column the amino acids are converted into keto acids and hydrogen peroxide under catalyzation of L- or D-amino acid oxidase. Hydrogen peroxide is detected amperometrically. The method has been tested by the analysis of beer, port, sherry, wine and fruit juice. A main emphasis was put onto the determination of D-alanine which can serve as an indicator for bacterial contamination. It is shown that a coupling of HPLC with enzyme reactors is a suitable technique for the rapid detection of this marker. Received April 14, 1999, Accepted September 15, 1999     

prebiotic transamination

Biological amino acids and alpha keto acids directly condense with decarboxylation and transamination to yield product amino acids. This process is closely related to unusual amino acid decarboxylase enzymes in certain microorganisms and may represent a primordial mode of amino acid metabolism.     

Formation of advanced glycation end (AGE) products in diabetes: Prevention by pyruvate and a-keto glutarate

Glycation of proteins and their subsequent structural and functional modifications have been ascribed to play a prominent role in the pathogenesis of several secondary complications of diabetes, such as cataract and retinopathy. In addition, it plays a role in the generalized ageing process as well. Investigations have been conducted to explore the possibility of preventing the above process by use of pyruvate and a-keto glutarate as representatives of physiologically compatible keto acids. The results demonstrate that both these compounds are effective in preventing the initial glycation reaction as well as the formation of AGE products. Both these compounds also inhibit the generation of high molecular weight aggregates associated with cataract formation. Mechanistically, the preventive effects appear to be due to (1) competitive inhibition of glycation by the keto acids and (2) the antioxidant (radical scavenging) properties of these compounds. The results are hence considered usefu l from the point of view of developing these and other keto acid derivatives as pharmacological agents useful in preventing glycation related protein changes and consequent tissue pathological manifestations.     

Measurement of alpha-keto acids in plasma using an amino acid analyzer

A method for measuring keto acid concentrations in physiological fluids using an amino acid analyzer was developed. After preliminary deproteinization and removal of amino acids, reduction with sodium cyanoborohydride at 105 degrees C resulted in efficient conversion of the keto acids to their corresponding amino acids. In applying the technique to plasma samples, the use of MeOH for deproteinization was necessary to avoid the large losses of keto acids that occurred during precipitation of proteins with perchloric acid. The method was used to follow plasma ketoisocaproate concentrations in rat plasma after administration of leucine, and was sufficiently sensitive to detect concomitant changes in other branched-chain keto acid concentrations.     

Relative importance of physiological precursors for ketogenesis in the nutritional homeostasis of the development of the embryonic chick

This paper investigates the relative importances of specific amino acids, and, in particular, branched chain amino acids and their keto derivatives as possible ketogenic precursors in suspensions of liver cells isolated from chick embryos. Addition of the branched chain keto acids stimulated ketogenesis. The order of potency was α-ketoisocaproic acid >α-ketoisovaleric acid >DL- α-keto-β-methyl-n-valeric acid. The relative order of effectiveness for branched chain keto acids was maintained at all comparable concentrations, and in each case maximum rates were observed with concentrations of 1–2 mM. In contrast to the stimulation of ketogenesis by their keto derivatives, branched chain amino acids were ineffective as precursors for ketogenesis. Of the other amino acids (utilised at concentrations present in chick embryo plasma) only Tyr, Lys, Phe and Arg produced significant increases in ketone body formation above the endogenous rate. At these physiological concentrations, the effectiveness of the amino acids were in the order of Tyr > Lys = Phe > Arg. The interactions between three groups of ketogenic precursor (fatty acids, amino acids and keto amino acids, all at physiological concentrations), produced rates of ketogenesis that were purely additive. These results indicate that high concentrations of hydroxybutyrate and acetoacetate found in plasma of developing chick embryos may arise from hepatic metabolism of several distinct precursors. The relative importance of each category of precursor may vary with the precise developmental status of animals.     

Biocatalytic asymmetric amination of carbonyl functional groups – a synthetic biology approach to organic chemistry

Transaminases catalyze amino transfer reactions from amino donors such as amino acids or amines to keto acids or ketones to give chiral amino acid or amines in optically pure form. α-Amino acid dehydrogenases catalyze the asymmetric reductive amination of α-keto acids using ammonia as amino donor to furnish L -amino acids. The distinct features and synthetic application of these two enzymes are reviewed in an effort to illustrate their promising and challenging aspects in serving as approaches to the direct asymmetric synthesis of optically pure amines from the corresponding keto compounds, a formidable problem in organic chemistry.     

Studies on the distribution and regulation of microbial keto ester reductases

In a limited screening 65 microorganisms were tested with regard to their ability to reduce keto acids or esters of different chain length and position of the keto group with NADH or NADPH as coenzymes. Twenty-seven organisms exhibited reductase activity. Among these, Candida parapsilosis and Rhodococcus erythropolis have been chosen for further investigation. The keto ester reductases of both C. parapsilosis and R. erythropolis prefer NADH as coenzyme and show higher activity towards keto esters than keto acids. The keto ester reductase production of C. parapsilosis during growth passed a maximum in the late exponential phase, decreased and reaches a plateau in the stationary phase. In contrast, the specific activity of the keto ester reductase of R. erythropolis did not decrease in the stationary growth phase. The enzyme of C. parapsilosis was inducible by a keto ester when growing on glycerol as the sole carbon source. Furthermore, the enzyme of C. parapsilosis was subject to catabolite repression. When C. parapsilosis and R. erythropolis were cultivated on n-alcane the specific activity of their keto ester reductases was enhanced about seven- and eightfold, respectively, compared to growth on glucose. This leads to the assumption that, while growing on n-alcane, a degradation product is formed in both strains that induces the production of the keto ester reductase. Correspondence to: M.-R. Kula     

An assay for transaminase B enzyme activity in Escherichia coli K-12

The Friedmann-Haugen assay for keto acids has been modified to allow the quantitative extraction of the hydrazone of a monocarboxylic keto acid from a mixture of hydrazones of mono- and dicarboxylic keto acids. The modification consists of extracting the monocarboxylic keto acid hydrazone into toluene as it is being formed. The extraction is performed in a tube, containing the reactants and toluene, held on a Vortex Mixer for 5 min. Using this assay to determine α-ketoisovaleric acid, the optimum conditions of pH and reactant concentrations for assaying the transaminase B activity in Escherichia coli were determined. The specificity of the assay for transaminase B in the isoleucine valine pathway is confirmed by results obtained with wild-type and mutant strains of Escherichia coli K-12 and Proteus mirabilis.     

Increased microsomal oxidation of hydroxyl radical scavenging agents and ethanol after chronic consumption of ethanol

The ability of the neonatal rat to oxidize the branched-chain amino acids leucine and valine and their corresponding keto acids was evaluated. In vivo, about 20% of orally administered labeled amino or keto acids were oxidized in 6 h, after which time little further oxidation occurred. In perfused neonatal liver the amino acids were oxidized at only 5-10% the rate of the keto acids. The oxidation of the keto acids showed a saturable dependence on concentration. The decarboxylation of ketoisocaproate (KIC) had a maximal rate of 40.1 +/- 1.6 mumol/h/g liver with an apparent Km of 0.27 +/- 0.03 mM, and decarboxylation of ketoisovalerate (KIV) had a maximal rate of 37.9 +/- 1.9 mumol/h/g liver and an apparent Km of 0.28 +/- 0.04 mM. KIC was ketogenic, producing mainly acetoacetate at a maximal rate of 44.5 +/- 1.6 mumol/h/g liver with an apparent Km of 0.27 +/- 0.03 mM. On the other hand, KIV was not gluconeogenic, although the perfused neonatal liver was able to produce glucose from lactate. During liver perfusion, KIV did not produce measurable quantities of either propionic or beta-aminoisobutyric acids, which are possible end products of KIV metabolism. Decanoic acid inhibited the decarboxylation of both keto acids to the same extent with a maximal effect at 0.4 mM fatty acid. At saturating levels, KIC was less ketogenic than decanoate. Inhibition of endogenous fatty acid oxidation by 2-tetradecylglycidic acid had no effect on keto acid oxidation. These data suggest that branched-chain amino acids derived from milk proteins are probably not quantitatively significant sources of either ketone bodies or glucose in the neonatal rat.     

AN EXAMINATION OF THE CATALYTIC PROPERTIES OF SEVERAL AMINOTRANSFERASES IN BRAIN AND LIVER BY MEANS OF AN IMPROVED RADIOMETRIC ASSAY WITH DINITROPHENYLHYDRAZINE

Abstract— The assay of aminotransferases, performed by solvent extraction of keto acids formed from labelled amino acids, has been modified to enhance the recovery of both aliphatic and aromatic keto acid products. The keto acids are first converted to their respective dinitrophenylhydrazones which are more completely extracted into less polar organic solvents. By this manoeuvre, both keto acid extraction is increased and the extraction of the precursor amino acid is reduced. Employing this technique, the kinetics of brain-stem γ-aminobutyric acid (GABA), tryptophan, 3,4-dihydroxyphenylalanine (DOPA) aminotransferases and brain-stem and liver tyrosine aminotransferases were examined. Brain-stem aminotransferases, particularly the aromatic amino acid transferases, have a higher affinity for both the amino acid and the keto acid when the aromatic keto acid, phenylpyruvate (0·8 mM), is employed as amino group acceptor, whereas maximal velocities for aminotransferase reactions are much greater when α-ketoglutarate (0·8 m m ) is the amino group acceptor. Brain-stem tyrosine aminotransferase exhibits a much lower affinity for tyrosine in the presence of either 0·8m m -α-ketoglutarate or 0·8 m m -phenylpyruvate than does liver tyrosine aminotransferase. p -Chlorophenylpyruvate and phenylpyruvate exhibit similar properties as amino group acceptors for brain-stem tryptophan aminotransferase. Cysteine inhibits tryptophan aminotransferase when phenylpyruvate is the amino group acceptor, in a manner which is competitive with the amino acid. Benzoylformate inhibits both tryptophan and DOPA aminotransferases when phenylpyruvate is the amino group acceptor, but this inhibition does not appear to be competitive with phenylpyruvate.     

Effects of abnormal metabolites of maple syrup urine disease on neurotransmitter receptor binding

The deficiency of keto acid decarboxylase in maple syrup urine disease results in the accumulation of branched chain amino acids and their corresponding keto acids in tissues and body fluids. The effects of abnormal metabolites were investigated on neurotransmitter receptor binding in rat brain. alpha-Keto acids caused selective in vitro decrease in alpha-adrenergic, beta-adrenergic receptor binding in synaptosomal preparations from rat brain. No significant changes were observed in binding of cholinergic, GABA, and dopamine receptors binding in appropriate rat brain preparations. These results indicate that selective inhibition of adrenergic receptor binding by branched chain keto acids may presumably account for neural abnormality in maple syrup urine disease.     

A gas-liquid-chromatographic procedure for separating a wide range of metabolites occuring in urine or tissue extracts

1. A gas–liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2°/min. being used over such part of the temperature range 30°–260° as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography–mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.